Glucans

ABSTRACT

The present invention relates to a glucan having a weight average molar mass of 15,000 to 50,000 g/mol on a single chain basis and a weight average molar mass in aqueous solution on an aggregate basis of 4 to 20×10 5  g/mol and existing in gel form in aqueous solution at a concentration ≧1% at 25° C. and neutral pH and having a melting temperature (gel to sol) of 30 to 44° C. when the glucan is dissolved in water at a concentration of 2%, methods for the production thereof, medical uses thereof, physical supports having the glucan applied thereto or impregnated thereon and in vitro methods of proliferation of skin cells which comprise contacting a population of skin cells with the glucan.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. patent application Ser. No.13/989,580 filed 9 Aug. 2013, which in turn is a filing under 35 USC 371of International Application No. PCT/GB2011/052357, filed 29 Nov. 2011,which claims priority to GB Application No. 1020190.3, filed 29 Nov.2010. Each application is incorporated herein by reference.

The present invention relates to a new glucan product, to processes forits manufacture and to uses thereof as a pharmaceutical, incorporated ina medical device, as a nutraceutical, cosmetic product or the like.

Glucans are a heterogeneous group of glucose polymers found in amongstothers the cell walls of plants, bacteria, fungi and protozoa. Glucanshave a backbone chain and in some cases side chains which, depending ofthe origin of the glucan, comprise β(1,3), β(1,4) and/or β(1M-linkedglucosyl units. Depending upon the source and method of isolation,beta-glucans have various degrees of branching and type of linkage inthe backbone and side chains. The frequency and type of linkage in theside chains is highly relevant to the molecule's biological activity.Glucans also differ highly in their molecular weight as well as in theirtendency for chain aggregation which both are essential features for theefficacy profile of these molecules. Most beta-glucans of fungal andyeast origin are in their native state insoluble in water, but can bemade soluble either by acid hydrolysis or by derivatization introducingforeign groups like-phosphate, -sulphate, -amine, -carboxymethyl and soforth to the molecule.

In Europe, Asia and USA, beta-glucans especially from Bakers' yeast havelong been employed as feed additives for animals, in cosmetics, asdietary supplement for humans, as immunomodulators e.g. in treatment ofwounds, and as an active ingredient in skin cream formulations. Glucanshave been employed in the treatment of cancer as shown in WO02/058711.Beta-glucans are, in this context, regarded as immunostimulantsincreasing the activity of white blood cells partly by inducing wellregulated and site restricted inflammatory reactions localised to thecancer. Their use in the treatment of inflammatory bowel disease hasalso been described in WO 2009/063221. Further applications of glucanswithin wound treatment are described in EP 815144 and in U.S. Pat. No.6,875,754 as well as for the treatment of asthma and allergy asdescribed in U.S. Ser. No. 12/528,215.

Cereal glucans comprise generally unbranched chains of β(1,3) and asignificant share of β(1,4) linkages while yeast glucans are made up ofpredominantly β(1,3) linked glucosyl residues with β(1,6) linkagesacting as branch points for side chains which may comprise both β(1,3)and β(1,6) linked glucosyl residues. Other molecules classed as glucansinclude curdlan, a basically linear molecule made up of β(1,3) linkedglucosyl residues without branches. Lentinan is a glucan with a β(1,3)linked backbone but incorporating single β(1,6) linked glucosyl residuesattached essentially regularly to the backbone giving a haircombstructure of this molecule. The single β(1,6) linked glucosyl residuesattached to the backbone equivalent to a β(1,3,6) linkage point but nofurther molecules are attached to this linkage point and thus glucanslike lentinan do not have side chains. Other examples of this group ofglucans are scleroglucan, laminarin and schizophyllan.

Variations in branching and the length and structure of the side chainslead to contrasting secondary and tertiary structures and thusbiological activities. The higher order structures of glucans varyconsiderably and molecular weight, solubility and particle size will allinfluence activity in a generally unpredictable manner. Some productsare extremely potent inducers of inflammatory cytokines in target cells,whereas others have the opposite effect, completely inhibiting cytokinerelease. Typical for many insoluble beta-glucan products is theinduction of a whole range of inflammatory responses, where e.g.injection of insoluble beta-glucan formulations has been associated withgranuloma formation, arthritis induction and increased susceptibilityagainst gram negative sepsis. On the other side, soluble beta-glucansare not reported to be encumbered with such negative side effects, buttheir efficacy as immunostimulants have been known to varysubstantially.

It has been shown (WO 95/30022), for example, that a glucan productderived from yeast which has been modified by glucanase treatment toselectively remove (1,6) linked side chains is more potent instimulating the immune system of fish than a product with intact (1,6)linked side chains.

Glucans have great potential as therapeutic agents and adjuvants but thevast range of structural variability, problems of analysis with suchlarge and complex molecules and the lack of understanding aboutmechanism of action and receptors for these molecules, means that thereis still a great need for an improved glucan product and forcontrollable and repeatable processes for manufacture of homogeneousproducts. The present invention addresses these problems. The presentinvention potentiates glucan efficacy by manipulating the primary andsecondary molecular structure of a glucan to establish apharmaceutically beneficial tertiary structure in the final product.

Beta-glucans are known to be so-called Pathogen Associated MolecularPatterns as they are found at the surface of a number of pathogenic(micro)organisms, especially fungi. Higher organisms have thus evolvedmechanisms for recognizing these types of structures in order to findand destroy intruders belonging to this class of organism. In mammalsthe so called innate immune cells express specific receptors recognizingbeta-glucans, and one of the most prominent receptors is calledDectin-1, but other receptors are also involved in the recognition orsignal transduction induced by beta-glucans amongst these are CD11b/CD18(CR3), and toll receptors 2 and 4 (TLR2 and TLR4). Of the cells involvedin recognizing beta-glucans are the typical phagocytes of the innateimmune system, i.e. monocyte, macrophages, dendritic cells, andgranulocytes, but also Natural Killer cells as well as a number ofendothelial cells and other more tissue specific cells have the abilityto express beta-glucan receptors.

The crucial step in inducing a biological response in the target cellsis the initial binding to the receptor and furthermore, it seems, theability of the beta-glucan formulation to cross-link a sufficient numberof receptors in order to induce an adequate signal-transduction into thecell. The present invention describes a product and a method for makinga product that has the ability to cross-bind receptors inducing aspecific type of biological activity. This is in contrast to insolubleproducts that could induce a massive response by cross-binding a largenumber of receptors and secondly be phagocytosed, which due to thenature of the insoluble (or “crystalline like”) glucan leads tolysosomal rupture within the cell inducing NLRP inflammasome activation.Insoluble beta-glucans may also induce ROS (reactive oxygen species)that also would trigger inflammasome activation leading to anunfavorable inflammatory reaction. The current invention describesbeta-glucans products that are able to induce a significant inflammatoryresponse that would activate several immune mechanisms, but withouttriggering inflammasome activation that is typical for a number of(aggregated insoluble) beta-glucan products.

The present invention potentiates glucan efficacy by establishing apharmaceutically beneficial supramolecular structure in the finalproduct.

The importance of higher order structure amongst β-glucans and thecontribution of the character of both individual glucan strands orchains and the higher order structure to the overall activity of theglucan product is described by Sletmoen et al. in Biopolymers vol. 89,No. 4 pp 310-321, 2008. Higher order structure may comprise a regulararrangement such as a triple helix or a more loose aggregation.

The present invention provides a glucan formulation that is perceived asa moderately sized entity when encountered by the target cells, but whenphagocytosed the glucan is easily taken up into phagosomes withoutinducing lysosomal rupture. The present invention thus describes a novelorganization of a highly potent soluble beta-glucan with good gellingproperties. Without wishing to be bound by theory it seems that theglucan molecules are arranged in a type of higher complex and loose“haystack” arrangement kept together by relatively weak hydrogen bondsbetween the frequent —OH groups along the glucan backbone structure. The“haystack” organization has the potential of presenting a number ofsites on its surface available for recognition by specific glucanreceptors on the target cells. The “haystack” organized molecules donot, however, harbor the rigidity of an insoluble product, but wouldmuch more easily become “degraded” and thus “immobilized” at the site orafter phagocytosis. Such a large higher order organization isadvantageous as compared both to insoluble and to known soluble productssince it gives an immunomodulatory response mimicking many of theeffects observed with particulate and insoluble beta-glucans withoutinducing less controllable and possible harmful effects known to beassociated with insoluble beta-glucans.

In one aspect the present invention provides a glucan having a weightaverage molar mass on a single chain basis of 15,000 to 50,000 g/mol anda weight average molar mass in aqueous solution on an aggregate basis of4 to 20×10⁵ g/mol, said glucan existing in gel form when dissolved inwater at a concentration ≧1% at 25° C. and neutral pH and having amelting temperature (gel to sol) between 30 and 44° C., preferably about33° C. when the glucan is dissolved in water at a concentration of 2%.The weight average molar mass values may conveniently be determined bySEC-MALS-RI analysis.

Preferably the glucan is in aqueous solution at a concentration of 1.5to 6%, more preferably 1.5 to 5%, still more preferably 2 to 4%, mostpreferably about 2%. It is understood that a “gel” form can beconsidered an aqueous solution.

In a preferred aspect the glucan is a beta glucan, preferably it has abackbone of β(1,3) linked glucosyl residues and side chains of β(1,3)linked glucosyl residues (e.g. side chains of at least 2, 5, 10 or 20linked glucosyl residues) attached thereto via a β(1,6) linkage.

“Neutral pH” means pH 7.

A “single chain” refers to an individual glucan molecule, i.e. one inwhich the glycosyl residues are covalently linked. “Aggregates” formthrough hydrogen bond interactions and define a supramolecular or higherorder structure. Such associations are less permanent than provided bycovalent bonding but the methods described herein result in recognisablepatterns of aggregation, whose average molar mass can be analysed usingthe techniques referred to herein. The “aqueous solution” is typicallypH 7.

Alternatively viewed, the present invention provides a gel glucanproduct comprising glucan in aqueous solution at a concentration of 1 to6%, the glucan having a weight average molar mass on an aggregate basisof 4 to 20×10⁵ g/mol and a weight average molar mass on a single chainbasis of 15,000 to 50,000 g/mol, the gel glucan product having a meltingtemperature (gel to sol)) between 30 and 44° C., preferably about 33° C.

As mentioned, the gel glucan product has a melting temperature (gel tosol) between 30 and 44° C., preferably about 33° C. when the glucan isdissolved in water at a concentration of 2%. It will be appreciated thathigher melting temperatures may be achieved by the inclusion ofadditional agents in the product, for instance gelling agents and/or byusing a higher concentration of glucan.

Glucan products are usually particulate, semi-soluble or in some casescompletely soluble in aqueous solutions, the latter either giving afluid clear solution as described, for example, in U.S. Pat. No.5,322,841 or some giving a viscous solution as described in Steiner etal (Frog Colloid Polymer Science 77, 1988) True gel forms of solublebeta-glucans are unusual, especially for soluble yeast glucans, but thepresent gel product has been found to provide excellent biologicalactivity, in particular in wound healing, as compared to other glucanproducts. In wound healing it is of utmost importance to apply apharmaceutical or medical device in a manner which secures themoisturization of the wound and the products must cover and stick to thewound surface to avoid infections and provide for an administrationprofile as deemed relevant by a medical practitioner or necessary due tothe type of wound. Usually, glucans in their particulate, semi-solubleor liquid form do not meet these basic requirements either because theyare not effective, they are in a state which is not applicable for woundhealing purposes, or both. The glucan of the present invention combinesthese necessary characteristics thus making it useful for allapplications where a pure glucan gel may find a proper use. In additionto strictly topical applications, other possible uses could be oraland/or mucosal administration, such as treating diseases of thegastro-intestinal tract or the oral cavity in addition to cancertherapy. The excellent adhesion properties of the glucan according tothe present invention enable it to cover the mucosal lining at the siteof action and thus accelerate the healing process. Thus the glucans ofthe invention have particular utility in the treatment of oral mucositisand other indications affecting the mucosa.

According to the present invention a radical heating and cooling processis performed to establish and “freeze” a preferred 3-dimensional complexand continuous glucan structure. This heating and rapid coolingestablishes a gel network with a very beneficial 3-dimensional structureof the glucan chains, which shows an excellent healing profile asexemplified herein. The tertiary, or 3-dimensional, structure of a betaglucan, in this case the arrangement of the molecular chains within theglucan gel as a whole, appears to be of utmost importance for efficacy.Without establishing a limitation of being bound by theory, it seemsthat only biologically effective molecular structures provide forbinding to different receptors at the target cells. Single chain, shortchain or products not structured in an appropriate 3-dimensional complexmanner will not be able to stimulate the body's immune system in thesame way.

There are limited ways to characterize the 3-dimensional (also definedas tertiary or supramolecular structure) molecular structure of a gelcomprised by its single chains. General ways of describing such a gelcan be by the average molar mass and molar mass distribution of thesingle chains, as well as by physical characteristics such as viscosity.In the case of immunomodulating products, gels can also be indirectlydescribed by their biological efficacy profile, or in other wordsmeasuring of the so-called “biological fingerprint”. When usingmolecular mass as a defining physical characteristic, it is recognisedthat the analysis methods are generally destructive, leading to theanalysis of the single chain components of the gel product, or smalleraggregated structures, rather than giving a detailed picture of themolecular interactions between these single chains which are necessaryto give a biologically effective 3-dimensional tertiary structure.Nevertheless a detailed analysis of several other physicalcharacteristics of glucans including their viscosity combined with abiological efficacy profile will enable the skilled man to distinguishbetween a variety of different glucans. One of these criteria is aspecific molecular mass range. The molar mass of glucans can bedetermined in different ways. In the case of a soluble glucan productthe molar mass is conveniently measured by SEC-MALS-RI analysis, andsuch analysis provides a weight average molar mass value (M_(W)) for thesample as well as the distribution of different molecular weights withinthe sample. In the present invention, the weight average molecular mass(M_(w)) is defined as follows:

$M_{w} - \frac{\sum{n_{i}M_{i}^{2}}}{\sum{n_{i}M_{i}}} - \frac{\sum{c_{i}M_{i}}}{\sum c_{i}}$

Where n_(i) is the number of molecules with molar mass M_(i). The weightconcentration c_(i) of molecules with molar mass M_(i) is proportionalto the molar mass M_(i) and the number of molecules n_(i).

c _(i) =M _(i) n _(i) =>n _(i) =c _(i) /M _(i)

The weight concentration for each slice in the chromatogram isdetermined by the RI-detector, while the molar mass for each slice ismeasured by the MALS-detector in combination with the RI-detector. Thecalculations are based on light scattering theory.

Specifically, the average molar mass (for single chains) according tothe present invention is determined by SEC-MALS-RI in DMAc with 0.5%LiCl (dimethylacetamide with 0.5% lithium chloride) assuming a do/dc of0.12 for the glucan in this solvent. The DMAc/LiCl solvent fullydissolves the said glucan into single chains, and subsequent SEC-MALS-RIanalysis with DMAc with 0.5% LiCl as eluent therefore gives a measure ofthe molecular weight distribution on a single chain level. In short, theanalysis of the glucan in DMAc/LiCl involves dissolution of the dryglucan in the solvent at a concentration of approximately 3 mg/ml bystirring the solution at r.t. over night and heating it at 100° C. for 1h, prior to the analysis by SEC-MALS-RI using 3× PLgel Mixed-A LScolumns and DMAc with 0.5% LiCl as eluent. The weight average molar massfor the glucan of the present invention on a single chain basisdetermined by this method is 15,000 to 50,000 g/mol, preferably 25,000to 45,000 g/mol, and more preferably 30,000 to 40,000 g/mol.

In aqueous solution the weight average molar mass of the mainly higherorder structures and aggregates present is 4-20×10⁵ g/mol, preferably5-15×10⁵ g/mol, and more preferably 6-12×10⁵ g/mol. These averages arecalculated when very large aggregates, i.e. molar mass above 1.0×10⁷g/mol, are excluded. The analysis of the glucan in aqueous solutioninvolves diluting the gel solution to approximately 3 mg/ml in 0.1 MNaNO₃ with 0.02% NaN₃, heating to 100° C. in a capped glass tube for 30min, cooling to room temperature, filtrating through a 0.2 μm syringefilter, and analysis by SEC-MALS-RI using TSKgel G5000 PWXL+TSKgel G4000PWXL columns and 0.1 M NaNO₃ with 0.02% NaN₃ as eluent. Similar set-upswith for example 0.05 M Na2SO4/0.01 M EDTA as solvent/eluent givesequivalent results. The combination of molar mass values for the singlechains and the higher order structures/aggregates in aqueous solutiongives a good indication of the molecular and tertiary structure of thegel as a whole and usefully defines the glucans of the presentinvention.

The glucans of the present invention are further characterized by beingin gel form at 25° C. in aqueous solutions with minimum concentration of1% and at a pH between 3 and 8. The glucan gels of the invention arefurther characterised by their viscosity profile exemplified by themelting temperature of the gels (gel to sol) of from 30 to 44° C.,preferably above normal body temperature, more preferably between 39 and44° C.

The gel melting point for a glucan product, i.e. the gel→sol transitiontemperature, is conveniently determined by small strain oscillatorymeasurements using a Stresstech HR rheometer or similar and examiningthe viscoelastic changes during cooling (70→10° C.) and heating (10→70°C.) of the glucan solution. An example of storage modulus (G′) plottedagainst temperature in such an experiment is shown in FIG. 3. Themelting temperature for this particular sample is equivalent to wherethe storage modulus of the curve for increasing temperature levels out(at approx. 0 Pa), which is approx. 33° C. Another way of determiningapproximate melting temperature of the gel is to measure the viscosity(e.g. using a rotational viscometer) of the gel at sequentially highertemperature until the viscosity is essentially gone and the gel hastransformed into a solution. The melting temperature is preferably about30-44° C., preferably over body temperature to guarantee a stabilizedglucan gel for topical applications. Topical administration demands acomparably lower melting temperature than oral administration oradministration to a site of an infection.

The glucan gel of the present invention is an aqueous gel and while thegel form can be confirmed by visual inspection, the non-newtonianviscosity profile and the pseudoplastic and thixotropic nature of theglucan gel may also be determined by viscosity measurement e.g. by usinga rotational viscometer. A 2% glucan gel according to the presentinvention has a viscosity of at least 1000 cP, preferably at least 1500cP, measured at 25° C. and a rotational speed of 10 rpm using aBrookfield DV-II+ Pro Programmable viscometer with a small sampleadapter and spindle SC4-31 (corresponding to a shear rate of 3.40sec⁻¹). A convenient method for measuring the viscosity of thispseudoplastic and thixotropic gel is to use a so called up-down rateramp, for example starting at 2 rpm and going up in 2 rpm increments to10 rpm and then going back down again in 2 rpm steps. The data from suchan experiment can both demonstrate the pseudoplastic (decreasingviscosity with increasing shear rate) and thixotropic (decreasingviscosity over time while subjected to shear) characteristics of the gelas well as provide a measure of e.g. 10 rpm viscosity. An example ofsuch data for a 2% glucan gel is shown in FIG. 6.

The glucans of the present invention are typically derived from yeast,preferably form Saccharomyces cerevisiae. The basic molecular structureof these glucans is typically a β-1,3-backbone (meaning a chain ofglucose molecules linked by β-1,3 linkages), in addition to β-1,3 sidechains (meaning a chain of at least two glucose molecules linked byβ-1,3 linkages) and a β-1,3,6-linkage point linking the side chains tothe backbone. In addition, glucans from yeast comprise β-1,6 linkageswhich may be linked to the side chains or directly to the backbone.Further types of linkages do exist but at a comparably low level. Otheryeasts which may provide a source for the glucan include Brewers yeast,Candida sp. like Candida albicans, Candida cloacae, Candida tropicalis,Candida utilis, Hansenula sp. like Hansenula wingei, Hansenula arni,Hansenula henricii and Hansenula americana, Histoplasma sp., Kloeckerasp., Kluyveromyces sp. like Kluyveromyces lactis, Kluyveromycesfragilis, Kluyveromyces polysporus, Pichia sp., Rhodotorula sp.,Saccharomyces sp. like Saccharomyces delbruekii, Saccharomyces rosei,Saccharomyces microellipsodes, Saccharomyces carlsbergensis or differentSaccharomyces strains like Saccharomyces cerevisiae R4 (NRRL Y-15903)and R4 Ad (ATCC No. 74181), Schizophyllum sp., Schizosaccharomyces sp.like Schizosaccharomyces pombe, Torula sp. and Torulopsis sp.

However, the gel glucans of the present invention may be derived fromother suitable sources, e.g. bacterial, fungal or cereal glucans. Thetherapeutic activities of various glucans are well documented in the artand the processes of the present invention may be used to enhanceactivity of glucans in general, in particular in wound healing where thephysical form and inter-molecular structure of the glucan product hasbeen shown, by the present inventors, to be particularly significant.Without wishing to be bound by theory a rule of thumb is that the higherthe weight average molar mass on a single chain basis of the glucan usedaccording to the present invention, the more efficacious glucan gels maybe produced.

The side chains of the glucan gels of the present invention usuallycomprise 2 or more β(1,3) linked glucosyl units. According to thepresent invention, single molecules linked to a main chain are notregarded as “side chains”.

The glucans of the present invention preferably have side chains of,i.e. consisting or consisting essentially of, β(1,3) linked glucosylunits. In addition to the β(1,3) linked side chains, the glucans mayalso have one or more β(1,6) linked side chains. By altering the chainsof the structure it is possible to alter the characteristics of thefinal product. There are many different ways of altering glucansincluding enzyme-treatment, use of acids like formic acid orhydrochloric acid or different bases as well as by other means.Preferred glucans are those which have been treated by acid (e.g. formicacid) or enzyme or any other suitable method to significantly reduce oreliminate the number of repetitive (1,6)-linked glucose molecules withinthe glucan. These (1,6)-linked glucosyl moieties would normally be foundin the side chains of beta-glucans derived from yeast. The resultingglucans have β(1,3) main chains and β(1,3) side chains which are linkedthereto through a single β(1,6) linkage which is not cleaved off by theelimination treatment.

The preferred glucans are essentially free of repetitive β(1,6) linkedglucosyl residues. The single (1,6) linkages at the branch points (theβ(1,3,6)-branching points) do not provide ‘repetitive’ β(1,6) linkedglucosyl units. By ‘essentially free’ is meant less than 6%, preferablyless than 4% and most preferably less than 3% of the total glucosylunits.

Some treatments, such as enzyme treatments, may leave up to 4beta-1,6-linked, but typically 2 beta 1,6 linked glucosyl unitsuncleaved in the side chains. Such molecules are also ‘essentially free’of repetitive beta 1,6-linked glucosyl units.

The distribution of linkages within preferred glucans of the inventionmay be represented as follows:

Type of linked glucosyl residue % β(1,3) 80-98 β(1,6) 0-6 β(1,3,6) 1-8Terminal 0.01-6  

β(1,3,6) refers to branch point residues which are (1,3) linked in thebackbone and participate in a (1,6) connection to provide a side chain.

The glucan of the present invention could be in the form of a single,extracted fraction or two or more different fractions with differentaverage molecular weights.

The glucans are underivatized in terms of chemical modifying groups.

The glucans of the invention are generated by a novel process. Theinventors have found that by performing a specific heating and coolingstep a novel gel glucan product is obtained with improved activity ascompared to other similar glucan products. By doing this a highlyrandomly organized “haystack” gel will be created without having thetypical triple helical structure of “annealed” beta-glucan chains.Surprisingly it was observed that this type of gel-structure wassignificantly more potent as immunomodulator than a classical organizedsoluble beta-glucan either in triple helical conformation or multiplesof helixes. According to this heating and cooling step, a solubilisedbeta-glucan preparation is energized in order to break up existinghigher order structure and inducing a random organization with a largeproportion (e.g. >40%, preferably >50%, more preferably >60% or >70%) offree single chain molecules

By rapid cooling in accordance with the present invention, the moleculesare “frozen” to a new molecular conformation by rapidly establishingintermolecular interactions wherein the product does not primarily formtriple helical structures. The molecules are thus frozen in a morerandom molecular position creating a novel intermolecular organisation.This supramolecular organisation is then resulting in a final productwhich is in a gel form. Surprisingly these new products have a muchbetter efficacy profile as immunomodulators compared to those not havingundergone this treatment or products without such a gel structure.

Thus in a further aspect the present invention provides a method ofproducing a gel glucan product as defined above wherein an aqueoussolution of glucan molecules is heated to a temperature of 120-130° C.,preferably 120-125° C., and held at that temperature for 10-30 minutes,the glucan solution is then cooled to a temperature of 35-50° C.,preferably 35-40° C., over a time period not greater than 80 minutes,preferably less than 60 minutes, e.g. 50-60 minutes.

The duration of cooling described above is based on a commercial processin which 220 litres of an aqueous solution of glucan molecules are usedas the starting material. It will be appreciated that if smaller volumesare used then the duration of the cooling step may be shorter than thatdescribed above, for instance less than 50 minutes, e.g. 20 to 50minutes.

The heating is preferably performed in an isolated and agitated tanklarge enough to hold the entire batch of product, with a jacket orsimilar structure to enable the heating of the outside of the tank. Thebatch size, the capacity of the heating system, the volume to surfaceratio of the tank and the effect of the agitator should be balanced insuch a way that the whole batch may be heated to the specifiedtemperatures within a reasonable time period, while ensuring ahomogeneous heating of the whole batch. Alternatively the energizingstep may take place after the product has been filled in its finalcontainer, either by heating in an autoclave or by alternative forms ofenergizing, e.g. ultrasound or micro waves.

Thus in a further aspect, the present invention provides a method ofproducing a gel glucan product as defined above wherein an aqueoussolution of glucan molecules is treated with an energy source to disturbthe higher order structure between the glucan chains and then treated toallow rapid reestablishment of intermolecular interactions. As well asheating, suitable energy sources include ultrasound and micro waves. Inthe case of ultrasound and micro waves, it may be sufficient to allowrapid re-establishment of intermolecular interactions simply to ceaseexposure to the ultrasound or micro waves.

If the energizing step has been performed for the whole batch in a tank,the active cooling is preferably performed in the same tank, and willrequire the ability to use the jacket of the tank to cool the tanksurface. Again the batch size, the capacity of the cooling system, thevolume to surface ratio of the tank and the effect of the agitatorshould be balanced to allow cooling to take place within the specifiedtime, while ensuring a homogeneous cooling of the whole batch. Thisinitial cooling should be followed by the filling of product into finalcontainers, and subsequent cooling of the containers to roomtemperature. Preferably the cooling step is performed immediately afterthe heating step, i.e. immediately (in so far as is practical with theequipment concerned) after the glucan has been held at the elevatedtemperature for 10-30 minutes.

A suitable procedure for performing the heating and cooling steps in anindustrial process is described in Example 1.

If the energizing step has been performed in the final containers, thesecontainers should be cooled to room temperature within the time framedescribed above.

The heating and cooling step described above may be repeated, e.g. oncemore.

The concentration of glucan in aqueous solution prior to the heating andrapid cooling step is preferably 1.5-6%, more preferably 2 to 4%, mostpreferably about 2%. Preferably, the concentration of glucan in theglucan gel is about 2%, for instance 1.8% to 2.2%. Therefore, preferablythe concentration of glucan in aqueous solution prior to the heating andrapid cooling steps is also about 2%. The methods of the presentinvention do not exclude the presence of further steps in whichadditional agents or materials are added to the solution. If such stepsare performed then the they may increase the volume of the aqueoussolution and so decrease the concentration of glucan in the solution.Preferably however the volume of the solution is not changedsignificantly so that the concentration of glucan in the starting andend products is roughly equal. Of course, the skilled man willappreciate that, if desired, a higher concentration of glucan in thestarting product can be used such that the addition of agents ormaterials in additional steps leads to a precise, desired glucanconcentration in the final product. The skilled man will be able tocalculate the appropriate glucan concentration in the starting productand the appropriate volumes of agents and materials to add to achieve adesired glucan concentration in the resulting gel product.

The above heating and cooling step may be performed on any aqueoussolution of glucan molecules; preferred glucans, including glucans withmodified branching, are discussed above and the glucan solution willpreferably be a yeast glucan solution. The weight average molar mass(M_(w)) of the glucans in the starting solution is preferably high,preferably, on a single chain basis, the weight average molar mass ofglucans in solution is above 15,000, more preferably above 20,000, mostpreferably above 25,000 g/mol. Suitable methods for determining thesemass values are given above.

Glucans are generally extracted from their source material (e.g. fungi,yeast or cereal) in particulate form but methods of generating solubleforms from particulate glucans are known in the art and include acid oralkali treatments, such as the formolysis step described in WO 95/30022.Soluble glucan products from cereals like barley are available fromSigma Chemical. According to the present invention, a particulatestarting material, such as may be prepared by the protocol in Example 1of WO 95/30022, will preferably be solubilised by heating in formic acidfor at least two hours. Formolysis performed on particulate glucanstarting material may conveniently cause selective removal of any β(1,6)linked glucosyl side chains as well as solubilising the particulateglucan.

The methods of the invention also preferably comprise a preliminaryheating step, prior to the above described heating and rapid coolingstep, where the formic acid treated product is boiled (>100° C.) for atleast 30 mins. After the product has cooled it is preferably treated toremove particulate materials by regular methods know in the art e.g. bycentrifugation or filtration.

The particulate glucan which is treated to yield a soluble form forprocessing in accordance with the present invention is preferablyderived from cell walls, in particular yeast cell walls, which have hadthe protein components and other remnants like mannan and chitin removedtherefrom e.g by washing.

One example of a suitable particulate yeast glucan product is producedby Biotec Pharmacon ASA which is derived from Bakers Yeast(Saccharomyces cerevisiae) and known as NBG Cos®. Another example ofparticulate glucan raw materials are whole glucan particles like theproduct Imprime WGP™. NBG Cos® is a natural underivatized (in terms ofchemical modifying groups) particulate β(1,3)/(1,6) glucan,characterised by NMR and chemical analysis to consist of polymers ofbeta-1,3-linked D-glucose containing side-chains of beta-1,3 andbeta-1,6-linked D-glucose.

The visual appearance of preferred gel products of the present inventionis firm, opaque and whitish with a high adhesion capacity to othersurfaces.

In a further aspect the present invention provides a glucan productobtained or obtainable by any of the aforementioned processes.

The glucans of the present invention are potent therapeutic agents andin a further aspect the present invention provides the glucans asdescribed herein for use in therapy, in particular for the treatment ofconditions where a subject is in need of a systemic or local enhancementof the immune response, e.g. where there is tissue damage or infection.The glucans are of particular utility in assisting wound or ulcerhealing and in the treatment of oral mucositis and cancer or reducingtumour size.

In a further aspect the present invention provides therefore a method ofassisting wound or ulcer healing or treating oral mucositis in a subjectin need thereof which comprises administration to said subject of aglucan of the present invention as described herein.

Reference is made to “assisting” wound or ulcer healing because somewounds or ulcers will heal naturally and others may not but the glucansof the invention have been shown to accelerate wound and ulcer healing.In some cases, healing may not occur satisfactorily without treatment.An example for such a wound which demands treatment for healing isdiabetic foot ulcer. In this indication the patient develops woundsbased on the underlying cause which is diabetes. Due to the oftenuntreated underlying cause and the fact that these wounds are to befound on the feet of patients, these ulcers do not heal by themselvesand cause huge problems for the patient usually ending in amputation ofthe foot.

In a further aspect the present invention provides a method of treatingcancer or reducing the size of a tumour in a subject which comprisesadministration to said subject of a glucan of the present invention asdescribed herein. Preferably the glucan is administered orally.Preferably, the glucan is administered at a dosage of 5 to 200mg/kg/day, more preferably 20 to 100 mg/kg/day.

In a further aspect the present invention also provides a pharmaceuticalcomposition comprising a glucan in gel form as defined above and one ormore pharmaceutically acceptable diluents or carriers, preferably waterand optionally one or more physiologically acceptable stabilisers orfurther diluents or carriers. The compositions may conveniently beformulated into any topical dosage form. The topical dosage forms may becreams, lotions, solutions, gels, ointments, pastes, sprays, films, etc.

In some variations, the compositions as described herein are in the formof an ointment. The ointment base may be an oleaginous base, anemulsifiable base, an emulsion base, or a water-soluble base. In othervariations, the compositions according to the present invention are inthe form of a cream. The creams may be viscous liquids or semisolidemulsions, either oil-in-water or water-in-oil. The cream bases may bewater-washable, and contain an oil phase, an emulsifier, and an aqueousphase. In yet further variations, the compositions of the presentinvention are in the form of a lotion. The lotions may be formulated assuspensions of solids and contain suspending agents to produce betterdispersions. The compositions according to the present invention mayalso be formulated pastes. Pastes are semisolid dosage forms in whichthe active agent is suspended in a suitable base. Depending on thenature of the base, pastes are divided between fatty pastes or thosemade from a single-phase aqueous gels.

In some variations, the compositions form a film on the wound surface.To aid film formation, film forming agents such as, but not limited to,acrylic acid and its derivatives, polyacrylic and its derivatives suchas polybutylmethacrylate and polymethacrylic acid, polymethacrylate,ascorbyl palmitate, carbomer, carnauba wax, cellulose derivatives suchas cellulose acetate phthalates, rosca mellose sodium, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, ethylcellulose and related compounds, hydroxypropyl methylcellulosephthalate, hypromellose phthalate, cetyl alcohol and derivatives,microcystalline wax, poloxamer, polyethylene glycol, polyurethane,polyvinyl acetate, polyvinyl acetate phthalate, polyvinyl alcohol,silicone rubber and derivatives, shellac, triglycerides derivatives, andcombinations thereof are used.

The compositions can also include at least one film plasticizer agentthat may serve to soften the polymer film formed by the film formingagent so that it is sufficiently flexible to move with area of the bodyapplied without cracking or peeling.

In some variations, the compositions may be cast into a film prior toapplication to the wound or applied to the wound directly where theypolymerize in situ. A “spread-on” film polymerizes when applied to theskin and may be delivered as a cream or ointment from a tube, roll-on,spray, and the like. The film may be created by incorporating a siliconerubber, into the external phase. Upon mixing with the internal phase,the resultant emulsion is allowed to cure and provides a “spread-on”film, which polymerizes when applied to the wound. The emulsion may bespread onto a substrate to achieve a desired thickness.

In other instances, the compositions may be preformed into a layer orpatch. The patch may be of varying thickness. The patch may also be cutto have a shape that generally follows the wound edges.

In some variations, the patches may include a pharmaceuticallyacceptable adhesive material that serves to affix the patch to the woundor skin. A patch backing layer may also be included.

When used as a spray, the compositions according to the presentinvention may include at least one organic solvent.

The compositions may be directly placed on a wound, or placed on asubstrate for application on a wound. Any substrate (carrier) may beused with compositions described here. For example, woven, non-woven,knitted, foam, and adhesive substrates may be used. Absorbent ornon-absorbent substrates may also be used. In some variations, thecompositions are sprinkled or spread on the substrate. In othervariations, the compositions are impregnated within the substrate.

The wound dressings may be applied for any suitable time period. Forexample, they may be applied over a time period of one day, over severaldays, over several weeks, or for several months or more. In general, thewound dressings will be reapplied until the wound is healed. Theduration of wound treatment with the dressings described here may dependon such factors as the type of wound being treated, wound location, andform of the composition being applied. Depending on the form used, thecomposition may be removed with water, or wiped or peeled off the wound.

The compositions described here may be used to treat wounds resultingfrom any etiology. For example, the wounds may be due to burns,infections, ischemia, lymphedema, neoplasms, neuropathy, radiationdamage, surgical procedures, venous insufficiency, and trauma. Thecompositions of the present invention are of particular utility inassisting wound or ulcer healing.

The invention further provides a physical support, for example anymedical device or material for medical use having applied thereto,including impregnated therein, a glucan of the invention as definedherein.

One important characteristic of such beta glucans is their water holdingcapacity and gel formation characteristics even in the absence ofconditions like non-neutral pH or cations which might promote gelhealing. Some beta-glucans would form gels at concentrations as low as1%, but more typically in the range of 2-4%. A soluble beta-glucan fromyeast like the one described herein will form a thixotropic andpseudoplastic gel when dissolved in aqueous solution at a concentrationof 1-6% in pH range from 3-7, independent of the presence of cations.

The compositions of the invention preferably comprise 1.5-6% beta glucanin an aqueous solution, more preferably the composition comprises around2-5% glucan in an aqueous solution. The use of different concentrationsis dependent on the purpose and the different modes of administration.As a general rule, a yeast glucan as described above with aconcentration of more than 4-5% in an aqueous solution and free fromother stabilizing substances would result in a final gel product whichis difficult to manufacture due to its solid gel properties.

Encompassed by the terms ‘wound’ and ‘ulcer’ are surface wounds,surgical wounds, burns, open fractures, leg ulcers, apthous ulcers,diabetic ulcers and decubitus ulcers. Wounds may be as a result ofinjury, surgery or disease but all are characterised by a loss of dermalintegrity, the skin may be torn, cut or punctured and regrowth of theskin is required to seal the opening. The glucans of the presentinvention have been shown to accelerate wound closure. As shown in theExamples, efficacy can readily be demonstrated by measuring the size ofan open wound.

The compositions are preferably applied topically, e.g. as a gel,transdermal patch, lotion, ointment, cream etc. Compositions may beapplied daily, more frequently or less frequently, e.g. twice daily oron alternate days and for a duration as determined by a clinician or insome cases by the patient or other health advisor. The duration oftreatment will depend on the nature and severity of the wound or ulcerwith progress generally being readily determined by visual inspection.

Topical administration includes administration in the mouth andsuitable, gels, lozenges, pastes, sprays etc. for delivery to the oralmucosa are known in the art.

The glucans and compositions containing them find utility in human andveterinary medicine. As used herein, the term ‘medical’ includesveterinary applications and contexts. Humans are preferred subjects fortreatment but other animals which may usefully be treated includelivestock and companion animals.

The glucans of the invention and compositions containing them may beapplied to or incorporated in a physical/solid support such as a patch,dressing, plaster, bandage, film, gauze etc. which can be applied to thewound or ulcer site and such products constitute a further aspect of thepresent invention.

The glucans of the present invention also find corresponding utility inin vitro applications for the culturing of skin cell lines, e.g. for usein skin grafts. Thus in a further aspect the present invention providesan in vitro method of proliferation of skin cells which comprisescontacting a population of skin cells with glucans of the invention asdescribed herein.

It will be appreciated that preferred features applicable to one aspector embodiment of the invention apply, mutatis mutandis, to all aspectsand embodiments.

The glucans of the present invention have excellent in vivo efficacy asanti-cancer agents and wound healing agents, as shown in the Examples.The Examples also show the ability of the glucans of the invention tostimulate production of cytokines which are relevant in a variety oftherapeutic contexts. The Examples show that the preferred glucans ofthe present invention trigger the expression of TNFα and CXCL2/MIP2α inmouse peritoneal macrophages. A weak induction, compared to the curdlanor LPS responses, of TNFα is also seen in human myeloid dendritic cellsderived from peripheral blood monocytes.

The effect of the preferred beta glucans on release of TNFα isdose-dependent and appears to diminish at glucan concentrations above acertain threshold value eg. 2-4 μg/ml in a variant of the RAW cell lineoverexpressing the beta glucan receptor dectin-1. A moderate to lowinduction of TNFα and CXCL-2 is special to the products of the presentinvention. Both TNFα and CXCL-2 are instrumental in wound healing. Themurine chemokine CXCL2 stimulates cell migration and angiogenesis, andcan be used as surrogate marker for angiogenic activity in theinflammatory granulation tissue.

The preferred glucans of the present invention do not trigger a powerfulexpression of IP-10 (CXCL-10). IP-10 is a member of the alpha orcysteine-X amino acid-cysteine (CXC) chemokine family of chemotacticcytokines. High levels of IP-10 expression have been detected in anumber of chronic human inflammatory conditions, including psoriasis, acommon inflammatory disease of the skin. Patients have generally shownan abnormal wound healing response characterized by a more intenseinflammatory phase and a prolonged and disorganized granulation phasewith impaired blood vessel formation. The glucans of the presentinvention should not enhance the LPS-induced expression of IP10 fromhuman dendritic cells, and preferably inhibit the LPS induced expressionof IP-10 from macrophages harvested from db/db mice. This shows that thepreferred glucans according to this invention turn on beneficialelements of the wound healing process while they turn off inhibitorsleading to a prolonged healing phase.

In addition, the Examples show the ability of the gel glucans of theinvention to activate the complement system as demonstrated byaccumulation of terminal complement complex (TCC).

The invention will now be further described in the followingnon-limiting Examples and the figures in which:

FIG. 1 illustrates the SEC-MALS-RI chromatograms of a number of batchesof branched β(1,3) glucan with <2% repetitive β(1,6) linked glucosylunits analyzed in DMAc with 0.5% LiCl assuming a dn/dc=0.12. As can beseen the molecular weight distribution is in the range of approx. 10,000g/mol to approx. 200,000 g/mol on the single chain level.

FIG. 2 shows SEC-MALS-RI chromatograms of a number of batches of theglucan product analyzed in aqueous buffer (0.1 M NaNO₃) assuming adn/dc=0.15. As can be seen the molecular weight distribution is in therange of approx. 10,000 g/mol to above 10,000,000 g/mol. The aqueousSEC-MALS-RI results, in combination with the results in DMAc/LiCl, showthat the glucan exist as aggregates in the aqueous solution.

FIG. 3 shows storage modulus, G′ (Pa), plotted against temperature for aglucan gel according to the present invention. The data was obtained bysmall strain oscillatory measurements using a Stresstech HR rheometerand the following temperature scan: 70 to 10° C. at a rate of ⅓° C./min,kept at 10° C. for 2 h and then 10 to 70° C. at a rate of ⅓° C./min. Themelting temperature of this gel (gel to sol) is determined toapproximately 33° C. based on where the increasing temperature curvelevels out (G′ 0 Pa).

FIG. 4 shows the viscosity measurements of a 2% glucan gel according tothe present invention using an up-down rate ramp method. The data wasobtained at 30° C. with an equilibration time of 3 min at 2 rpm prior tofirst measurement and 30 sec equilibration before measurement on eachconsecutive speed after that. Calculated 10 rpm viscosity was 1772 cPfrom the IPC Paste model in the Rheocalc software of BrookfieldEngineering Inc.

FIG. 5 describes the release of TNF-α from human myeloid dendritic cellsderived from peripheral blood monocytes cultured in the presence of 200μg/ml of the glucan gel of the present invention, curdulan or LPS. Thecytokine was measured in culture medium supernatants at 24 hpost-stimulation using a commercially available ELISA kit.

FIG. 6 illustrates the release of CXCL10 (IP10) from human myeloiddendritic cells derived from peripheral blood monocytes cultured in thepresence of 200 μg/ml of the glucan gel of the present invention,curdlan or LPS. The chemokine was measured in culture mediumsupernatants at 24 h post-stimulation using a commercially availableELISA kit.

FIG. 7 shows the secretion of CXCL-10 from macrophages harvested fromdb/db mice costimulated in vitro by LPS and the glucan gel of thepresent invention. Lane 1; LPS alone, lane 2; LPS+20 μg/ml of the glucangel of the present invention, lane 3; LPS+2 μg/ml of the glucan gel ofthe present invention. *p<0.05. *p<0.01.

FIG. 8 shows the results of a wound healing trial using the glucan gelaccording to the present invention in a 2% and 4% concentration in anaqueous solution compared to a growth factor cocktail with a knownefficacy profile. The dressing+water was used as vehicle control. Both2% and 4% concentrations are effective, with 4% being more effective,although less effective than the GF cocktail.

FIG. 9 shows activation of complement by different batches ofbeta-glucans. Accumulation of fluid-phase terminal complement complex inhuman serum was measured. 241-7, 231-0, 411-8, 391-8 are beta-glucans ingel form representing glucans of the present invention. VLMSG representa non-complement activating formulation of a soluble beta-glucan whichis not in gel form. The horizontal dotted line represents thespontaneous complement activation in human serum.

FIG. 10 shows secretion of TNFα from a dectin-1 over-expressing RAWcell-line variant. The soluble yeast beta glucan 421-4 represents aglucan of the present invention, while 161194, 30395, xx0995 and 51196are clear, non-gelling variants.

FIG. 11 shows the biological effect in the dectin-1 over-expressing RAWcell line of soluble glucan (SG) subjected to heating and rapid cooling(HC) as described in Example 1. GC065 rn 9270 is a soluble glucanproduct with a broken gel, as described in Table 1. Treatment accordingto Example 1 rescues the biological effect of the broken product.

FIG. 12 shows the biological effect in the dectin-1 over-expressing RAWcell line of soluble glucan (SG) subjected to heating and rapid cooling(HC) as described in Example 1. 421-4 is a SG product corresponding tothe product described as a soft gel in Table 1. Treatment according toExample 1 enhances the biological effect of SG batch 421-4.

EXAMPLES Example 1 Preparation of Gel Glucan Product of the PresentInvention

An aqueous solution of 1.5 to 2% yeast glucan molecules was treated asdescribed below. This aqueous solution was prepared from a particulateglucan preparation by formolysis to selectively remove β-1,6 side chainsand subsequent purification and diafiltration to remove particulatematter and low molecular weight components from the formolysis solution.A suitable formolysis step is disclosed in Example 3 of EP 0759089 B1.The particulate glucan was itself prepared from cell walls of Baker'sYeast (S. cerevisiae) by separate extractions with alkali, ethanol andwater, each extraction being followed by appropriate drying (spraydrying and vacuum drying).

a. Heat Treatment:

Heat treatment takes place after the concentration of the glucansolution has been adjusted, normally giving a product volume ofapproximately 220 liters at a temperature of approximately 60° C., in aclosed and agitated 800 liter tank which is heated by introduction ofsteam to a jacket surrounding the tank.

The product is heated slowly to approximately 105° C. to ensure an evenheating of the whole batch, and then more quickly to 123° C. Normalheating time from 60 to 123° C. is 40-50 minutes. The product is thenheld at 123-125° C. for 20 minutes.

b. Active Cooling:

Active cooling is then started. It is operated manually, by directopening and closing of hand operated valves. First the steam iscarefully evacuated from the jacket to drain, and the drain valves areleft open. Cooling water is then carefully introduced to the jacket,slowly at first to avoid excessive thermal stress to the steel of thetank. As the temperature drops the flow of water is increased. Coolingis normally continued until the product temperature reaches 35-40° C.Normal cooling time from 123 to 40° C. is 50-60 minutes.

Example 2 In Vivo Wound Healing in Mouse Model

The impact of test glucans and controls on wound healing wasinvestigated by analysing the repair of full-thickness excisional skinwounds in the diabetic (db/db) mouse model (i.e. BKS.Cg-mDock7^(m)+/+Lepr^(db)/J mice). Upon acclimation (5-7 days withoutdisturbance) the animals were housed in groups of 5 animals according toHome Office regulations and the specific requirements of diabeticanimals. After experimental wounding, animals were housed in individualcages (cage dimensions 35×15×15 cm with sawdust bedding, changed twiceweekly), in an environment maintained at an ambient temperature of 23°C. with 12-hour light/dark cycles. The mice were provided with food(Standard Rodent Diet) and water ad libitum. Following all anaestheticevents, animals were placed in a warm environment and monitored untilthey were fully recovered from the procedure. All animals receivedappropriate analgesia (buprenorphine) after surgery and additionalanalgesics as required. All animal procedures were carried out in a HomeOffice licensed establishment under Home Office Licences (PCD: 50/2505;PPL: 40/3300; PIL: 50/3482; PIL: 70/4934). The health of animals was illmonitored on a daily basis throughout the study.

On day 0, animals were anaesthetised (isofluorane & air) and the dorsumshaved and cleaned with saline-soaked gauze. A single standardisedfull-thickness wound (10.0 mm×10.0 mm) was created in the left dorsalflank skin of each experimental animal. Wounds in all treatment groupswere subsequently dressed with a circumferential band of the transparentfilm dressing Bioclusive™ (Systagenix Wound Management, UK); after whichthey received a glucan or control by injection 50 μl of a 2% solution inpurified water through the Bioclusive film using a 29-gauge needle.Diabetic animals were randomized to one of the treatment regimes usingappropriate software. For the experimental groups receiving glucantreatments was reapplied on post-wounding days 2, 4 and 6. Wound sitesin these animals were closely monitored for excessive build-up ofapplied agents and excessive wound site hydration; if excessive appliedagent accumulation/hydration was apparent, previously applied materialwas removed by aspiration prior to reapplication. For the positivecontrol group treatments was reapplied daily until post-wounding day6—wounds in this group received a total of 7 applications of the growthfactor combination treatment. On post-wounding days 4, 8 and 12 allanimals were re-anaesthetised, their film dressings and any free debrisremoved, and their wounds cleaned using saline-soaked sterile gauze.After photography on days 4 and 8, wounds were re-dressed as above withBioclusive film dressing. Healing was determined as wound closurerelative to the wound size at day 0.

The results are shown in Table 1 and FIG. 8 below.

TABLE 1 Treatment of wounds in diabetic mice Healing Healing Healingwounds, wounds, wounds, Product Day 8 day 12 day 15 Negative control(dressing only) 0/10 1/10  2/10 Vehicle control (water + dressing) 1/103/10  4/10 Broken gel product 3/10 7/10  7/10 Soft gel glucan (2%) 5/109/10 10/10 Solid gel glucan (4%) 8/10 10/10  10/10 Positive control(GFcoctail) 10/10  10/10  10/10

The soft gel glucan product and the solid gel glucan product are bothyeast derived glucans which have been prepared by the methods of thepresent invention. Both are yeast derived glucans which have beentreated with formic acid to remove β(1,6) linked glucosyl units found inthe native yeast glucan side chains. The solid and soft gel glucans havebeen prepared using the new heating and rapid cooling protocol describedherein (Example 1). Surprisingly the solid gel (a 4% product)demonstrates enhanced wound healing activity as compared to the soft gelglucan product (a 2% product).

The “broken gel product” describes a yeast derived glucan product wherethe (inter-molecular) conformation of the molecules in the gel has beendestroyed to a large degree by exposure to an agent which interfereswith hydrogen bonding. This glucan is not able to exert a similarbeneficial efficacy/healing profile compared to the gel product of theinvention. This result clearly shows that the gel structure of theglucan according to the present invention is a surprisingly importantproperty for in vivo efficacy.

The results clearly show that the use of a glucan gel produced accordingto the invention elicits a more potent response in wound treatmentmodels compared to the delivery vehicle and negative control. The factthat the “broken gel” product gives inferior results also points to thenecessity of the existence of an intact gel structure or specific highorder conformation within a glucan gel.

Example 3 Determination of Molar Mass

The molar mass of a series of yeast glucan products was determined usingsize exclusion chromatography as previously defined in the description.The experiment was performed with the glucan in aqueous solution andthus gives molar mass values for the glucan aggregates within the glucansample and not on a single chain bases. 5 glucan samples were tested,all were derived from yeast and all had had their β(1,6) linked sidechains diminished. 4 were in solution and the 5th was a gel glucan inaccordance with the present invention (prepared in accordance withExample 1).

The calculated average molar mass for the four glucans in aqueoussolution varied from 1.0-3.74×10⁵ g/mol.

The glucan of the invention had an average molar mass of 8×10⁵ g/mol.

In other experiments, the glucans of the present invention had molarmasses which varied from 5-15×10⁵ g/mol.

Example 4 Determination of Melting Point

Determination of the melting point of a glucan gel produced according tothe present invention was performed as described in the description andthe results are shown in FIG. 3.

Example 5 Viscosity Measurements

Determination of the viscosity of a glucan gel produced according to thepresent invention (prepared according to Example 1) was performed asdescribed in the description and the results are shown in FIG. 4.

Example 6 Biological Activity

The effect of the gel glucans of the present invention, which wereprepared as different batches, each according to Example 1, on therelease of TNFα and CXCL-2 and -10 is described herein and shown in theFigures.

In addition, the ability of the beta glucans to activate the complementsystem was measured. The complement system is composed of a series ofserum proteins. The system is a part of the innate immune system, and isactivated upon infection or detection of pathogen associated molecularpatterns. Activation of the system results in a cascade of cleavage ofthe complement proteins, which ultimately leads to formation of aterminal complement complex (TCC). Accumulation of TCC can be measuredby detection of a neo-epitope using monoclonal antibodies, and can thusserve as an indicator of complement activation.

The glucan was diluted (1:10) in human serum to a volume of 100 Themixture was incubated at 37° C. for 30 min, then diluted 1:5 in PBSbefore the relative amount of fluid-phase TCC was determined intriplicate using a commercially available ELISA-test kit for human TCC.The results are shown in FIG. 9.

The soluble beta-glucan of the present invention, in gel form, activatesthe complement system in human serum. However, activation of complementis not a common feature of soluble beta-glucans, as exemplified by VLMSGin FIG. 9. The molecular weight of VLMSG range from 10³ to approximately5×10⁶ g/mol, with a mean value 1,3×10⁴ g/mol, and yields a clearsolution.

It was demonstrated that gel-forming soluble yeast beta glucans of thepresent invention prepared according to Example 1 stimulate the releaseof TNFα from dectin-1 over-expressing RAW cells, while non-gelling,clear, variants of soluble yeast beta glucans apparently do so to a muchlesser extent (FIG. 10).

1. A gel glucan product comprising a soluble yeast glucan in aqueoussolution at a concentration of 1 to 6%, the glucan having a weightaverage molar mass of 15,000 to 50,000 g/mol on a single chain basis anda weight average molar mass in aqueous solution on an aggregate basis of4 to 20×10⁵ g/mol, the gel glucan product having a gel to sol meltingtemperature of 30 to 44° C.
 2. The gel glucan product of claim 1,wherein said glucan has a weight average molar mass of 20,000 to 40,000g/mol on a single chain basis.
 3. The gel glucan product of claim 1,wherein said glucan has a weight average molar mass of 25,000 to 30,000g/mol on a single chain basis.
 4. The gel glucan product of claim 1,wherein said glucan is in aqueous solution at a concentration of about2%.
 5. The gel glucan product of claim 1, wherein said glucan is derivedfrom Saccharomyces cerevisiae.
 6. The gel glucan product of claim 1,wherein said glucan is a beta glucan comprising a backbone ofβ-(1,3)-linked glucosyl residues and side chains comprising 2 or more(3-(1,3)-linked glucosyl residues, the sidechains being attached to thebackbone via a (3-(1,6)-linkage.
 7. The gel glucan product of claim 1,wherein said glucan is essentially free of repetitive β(1,6) linkedglucosyl residues.
 8. The gel glucan product of claim 1, wherein saidgel glucan product has a gel to sol melting temperature of about 33° C.9. The gel glucan product of claim 1, which is present in apharmaceutical composition also comprising one or more pharmaceuticallyacceptable diluents or carriers.
 10. A physical support having appliedthereto or impregnated therein the gel glucan product of claim
 1. 11.The physical support of claim 10, wherein said physical support isselected from the group consisting of a woven, non-woven, knitted, foamor adhesive substrate; a patch, dressing, plaster, bandage, film orgauze.
 12. A gel glucan product obtainable by a) heating an aqueoussolution of soluble yeast glucan molecules to a temperature of 120° C.to 130° C. and holding the solution at that temperature for 10 to 30mins; and b) cooling the glucan solution to a temperature of 35° C. to50° C., over a time period of less than 80 minutes, wherein said glucanis present in said gel glucan product at a concentration of 1 to 6%, hasa weight average molar mass of 15,000 to 50,000 g/mol on a single chainbasis and a weight average molar mass in aqueous solution on anaggregate basis of 4 to 20×10⁵ g/mol, and wherein said gel glucanproduct has a gel to sol melting temperature between 30° C. and 44° C.13. The gel glucan product of claim 12, wherein said glucan has a weightaverage molar mass of 20,000 to 40,000 g/mol on a single chain basis.14. The gel glucan product of claim 12, wherein said glucan has a weightaverage molar mass of 25,000 to 30,000 g/mol on a single chain basis.15. The gel glucan product of claim 12, wherein said glucan is inaqueous solution at a concentration of about 2%.
 16. The gel glucanproduct of claim 12, wherein said glucan is derived from Saccharomycescerevisiae.
 17. The gel glucan product of claim 12, wherein said glucanis a beta glucan comprising a backbone of β-(1,3)-linked glucosylresidues and side chains comprising 2 or more (3-(1,3)-linked glucosylresidues, the sidechains being attached to the backbone via a(3-(1,6)-linkage.
 18. The gel glucan product of claim 12, wherein saidglucan is essentially free of repetitive β(1,6) linked glucosylresidues.
 19. The gel glucan product of claim 12, wherein said gelglucan product has a gel to sol melting temperature of about 33° C. 20.The gel glucan product of claim 12, wherein in step a) the glucan isheld at the temperature of 120° C. to 130° C. for about 20 minutes. 21.The gel glucan product of claim 12, wherein in step a) the aqueoussolution of glucan molecules is heated to a temperature of 120° C. to125° C.
 22. The gel glucan product of claim 21, wherein in step a) theglucan is held at the temperature of 120° C. to 125° C. for about 20minutes.
 23. The gel glucan product of claim 12, wherein the glucan iscooled over a time period of 50 to 60 minutes.
 24. The gel glucanproduct of claim 12, wherein said method is preceded by a formolysisstep wherein a particulate glucan starting material is suspended informic acid in order to remove β-(1,6) linked glucosyl side chains andto solubilise the particulate glucan.
 25. The gel glucan product ofclaim 24, wherein the formolysed product is filtered through a mesh ofabout 0.2μ.
 26. The gel glucan product of claim 12, which is present ina pharmaceutical composition also comprising one or morepharmaceutically acceptable diluents or carriers.
 27. A physical supporthaving applied thereto or impregnated therein the gel glucan product ofclaim
 12. 28. The physical support of claim 27, wherein said physicalsupport is selected from the group consisting of a woven, non-woven,knitted, foam or adhesive substrate; a patch, dressing, plaster,bandage, film or gauze.